One of the most ravaging diseases of the late twentieth century has been AIDS (acquired immunodeficiency syndrome), brought on by HIV (human immunodeficiency virus) infection. Currently, there are no cures for this disease and minimally effective treatments. One of the problems that exist in the development of therapies for HIV infection is that the HIV virus rapidly develops resistance to a wide variety of chemotherapeutic agents. HIV, particularly human immunodeficiency virus type 1 (HIV-1), mutates over time to become resistant to many of the antiviral drugs administered for treatment. AIDS physicians need to know when the antiviral therapy being used to treat a individual patient is no longer effective so that the antiviral drug or drug combination can be modified, thereby minimizing viral replication and the onset of immunodeficiency symptoms.
Reverse transcriptase (RT) inhibitors such as zidovudine (ZDV, also referred to as azidothymidine (AZT)), didanosine (ddI or dideoxyinosine), zalcitabine (ddC or dideoxycytosine), lamivudine (3TC), stavudine (d4T), and nevirapine (NVP) are nucleoside or non-nucleoside analogs currently approved for the treatment of HIV-1 infections. 3TC is known to have potent anti-HIV-1 activity and minimal toxicity, and is one of the most commonly used drugs in combination therapy as first-line treatment for HIV-1-infected patients. 3TC administered in combination with AZT provides greater and more sustained increases in CD4+ cell counts, and higher reductions in HIV-1 RNA viral load than continued AZT or 3TC monotherapy. 3TC in combination with AZT and protease inhibitors slows the progression of HIV-1 disease and reduces levels of HIV-1 RNA to less than 500 copies per milliliter for as long as one year in 90% of patients (Gulick et al., N. Engl. J. Med. 337: 734-739 (1997)).
However, the use of reverse transcriptase inhibitors, such as 3TC, in both monotherapy or combination therapy has resulted in the emergence of drug-resistant variants of HIV-1 (Gulick et al., N. Engl. J Med. 337: 734-739 (1997)). For a drug such as 3TC, the resistance is conferred by mutations in codon 184 of the HIV-1 reverse transcriptase gene, which replaces the wild type methionine residue (M; ATG) with a valine (V; GTG) via a transient substitution with an isoleucine (I; ATA). The presence of this M184V mutation has been associated with a greater than 500-fold resistance to 3TC and the partial loss of the anti-retroviral and clinical benefits of the drug. It is therefore important to monitor for drug resistance in individuals treated with reverse transcriptase inhibitors.
Phenotypic assays provide direct and definitive evidence of resistance to reverse transcriptase inhibitor drugs. However, presently available assays for the analysis of phenotypic resistance are based on virus culture and are therefore labor intensive and time consuming (two to five weeks), costly, and unsuitable for rapid clinical monitoring or surveillance of drug resistance (Kavlick et al., Antiviral Research 28: 133-146 (1995); Wainberg et al., AIDS 9: 351-357(1995)). In addition, these assays are fraught with biologic variabilities, including those related to viral isolation and tropism. Since tissue culture is highly selective for viral strains with in vitro growth advantages, these culture-based assay methods may not be representative of the total virus population present in vivo (Li et al., J. Virol. 65: 3973-3985 (1991)).
In the absence of rapid phenotypic assays, genotypic tests are currently being used to provide indirect evidence of resistance. Genotypic testing monitors for the presence of mutations that are associated with resistance, such as the M184V mutation. Among these genotypic tests, primer-specific PCR, point mutation, and reverse hybridization assays are the most commonly used (Wainberg et al., AIDS 9: 351-357(1995); Frenkel et al., J. Clin. Microbiol. 33: 342-347 (1995); Stuyver et al., Antimicrob. Agents Chemother. 41: 284-291 (1997)). Unfortunately, clinical monitoring of reverse transcriptase inhibitor drug resistance by genotypic testing may not detect unrecognized mutations or potential synergistic or antagonistic effects of complex mutation patterns arising from combination therapy with different reverse transcriptase inhibitors. For example, the suppression of phenotypic resistance to AZT conferred by the M184V mutation clearly illustrates the effect that a combination of mutations may have in a given phenotype (Larder et al., Science 269: 696-699 (1995)). Also, genotypic testing only detects resistance associated with known mutations (i.e., codon 184 for 3TC resistance).
U.S. Pat. No. 5,631,128 to Kozal describes polymerase chain reaction (PCR) assays for monitoring antiviral therapies in the treatment of AIDS. These genotypic assays use PCR to measure HIV-1 RNA copy number in plasma or to measure specific known HIV-1 RNA mutations, namely the mutation at codon 215 or codon 74 of the pol gene. The HIV-1 RNA copy number is an indication of the circulating HIV viral load. A decrease in HIV-1 RNA copy number correlates with successful antiretroviral therapy, whereas an increase in HIV-1 RNA copy number indicates disease progression, most likely caused by resistance to therapy. Therefore, the genotypic assays described in U.S. Pat. No. 5,631,128 detect only previously identified viral RNA mutations and are incapable of detecting phenotypic resistance caused by known or novel mutations, or the assays detect a rise in HIV-1 RNA copy number, which could be due to conditions other than resistance. An incorrect diagnosis of drug resistance followed by cessation of the antiviral therapy being administered could result in exacerbation of a disease that had been responding to therapy.
Therefore, there is a need for sensitive, rapid methods for the detection of HIV resistance to drug therapies in patients so that, if the virus becomes resistant to a particular drug or combination of drugs, the therapy can be modified, thereby keeping viral replication to a minimum and preventing or postponing the onset of AIDS.